The present invention relates to methods and devices for pipetting aliquots of material, for example, samples and reagents, into multiwell receptacles such as, but not limited to, microtiter plates, especially using a mask with openings or holes in which the openings are adapted to allow the particular additions to be made into the receptacle. These masks or covers also serve as masks that reveal only certain wells in the multiwall receptacle at a given time. Use of these masks reduces the risk that operator error will result in incorrect addition of reagents, thereby compromising the validity of assay results. While current embodiments relate to SBS standard microtiter plates, the invention may be applied to other multiwell receptacle formats currently in use or which may be developed.
Real-time PCR (qPCR) has become a standard method for determining the relative concentration of molecules for a specific nucleic acid sequence within a complex population of sequences from a biological sample. It is one of many state of the art instrumentation systems that utilize SBS microtiter plates. Microtiter plates, in 96-well and 384 well formats, have become the standard reaction vessel format for qPCR, with 96-well being most prevalent. The SBS format has become a standard for many other assays than qPCR, and many instrument and automated systems have been developed based on use of disposable components adapted to the SBS format. Other formats have been and could be used to array multiple wells in a moveable device. In particular, a multiwell receptacle in which the wells are arranged in a circle can be used in automated systems, the Rotor-Gene Q system being an example. Since a circular arrangement of wells is symmetrical around the center of the circle, addition of reagents to the wells is especially prone to mistakes by manual operators. The present invention can be adapted to these other multiwell receptacle formats.
Traditionally for qPCR, the reaction wells in the plate are utilized so that a single plate has multiple samples and standards analyzed by one or a few target assays. Additional target assays are performed on additional plates primarily due to the large number of wells required for the standards. It is desirable to prepare reaction mixtures as partial pre-mixes to minimize the number and complexity of dispensing steps performed because these steps are most often done by manual pipetting. In the traditional format, the premix is prepared with all but the sample nucleic acids and dispensed into the portion(s) of the plate designated for that target assay. Each sample nucleic acid, up to 96 different ones per plate, is then individually dispensed into pre-assigned wells for the analysis.
There are two major disadvantages to this dispensing system. First, each sample is diluted into every reaction premix well for every target assay, thereby introducing multiple sample dilution errors both within sample replicates and across target assays. Because qPCR measures concentration in each reaction well, with the dispensed volumes of both the premix and the sample contributing to the total variance, there is the potential to increase the level of variation in data and significantly influence the relative concentration results. Second, this strategy requires that when multiple samples are analyzed, they all must be available for analysis at the same time. In other words, samples originating at different times must be stored until an appropriate number have been obtained to fill the capacity of the microtiter plate. Since an appropriate sample number is typically 10 to 100, an investigator is not afforded the opportunity to evaluate initial data early in the sample collection phase to determine whether there are problems with this process.
Recently a new qPCR plate utilization system has become popular that eliminates these disadvantages. This system utilizes the same basic approach as high density microarrays wherein a single sample is applied once to a large number of target assays arrayed on a single reaction platform, in this case the PCR plate. In this qPCR array format, the target-specific assay reagents, i.e. the PCR primers, are pre-dispensed individually into their assigned reaction wells on the PCR plate and dried into the bottom of the reaction wells. The nucleic acid from a single sample is diluted once into a reagent pre-mix containing all other components necessary for qPCR, thereby using only a single dilution step for all the assays. This uniformly diluted pre-mix with sample is dispensed into all the wells on the plate by repetitive pipetting utilizing a multi-channel pipette. Thus, the user has only one sample solution to dispense over an entire plate and can analyze a single sample for up to about 84 target assays on a single 96-well microtiter plate. Dispensing of the one sample across the entire plate can be viewed as the analogy of applying a single coat of paint color across a surface.
This latter format can be enhanced by the use of 384-well plates (24×16 well configuration) to consolidate four different 96-well array formats into a single higher throughput run. From a plate management perspective for dispensing the target specific primers, it may be easiest to divide the 384-well plate into four registers of 96-wells by alternating rows and columns on the 384-well plate (see FIG. 1 for well assignment map). This well assignment structure, while still compatible with standard 8- or 12-channel pipettors, requires the user to keep track of alternating well positions for each sample throughout the dispensing process. In keeping with the painting analogy, dispensing each register of wells represents one color and, as in painting, therefore masking to prevent incorrect application of color over a specified area can be desirable. Alternate formats of the masks can be envisioned, specifically sets of masks for the 384-well plates can also accommodate 8 subsections of 48 wells, 12 subsections of 32 wells, 16 subsections of 24 wells, 24 subsections of 16 wells, or 32 subsections of 12 wells. Also for 96-well plates, sets of masks can accommodate 2 subsections of 48 wells, 3 subsections of 32 wells, 4 subsections of 24 wells or 8 subsections of 12 wells.
The multiwell devices with which the mask(s) of the invention can be used can have spatial arrays corresponding to those of standard microtiter plates, such as E-well, 12-well, 24-well, 96-well, 384-well, or 1536-well microtiter plates in the Society for Biomolecular Screening (SBS) format or other formats commonly used in the industry. See, e.g., Journal of Biomolecular Screening, Vol. 6, No. 2, 2001, p. 61-68. Thus, the multiwell devices may be, and advantageously are, compatible with conventional microtiter plate-related products, such as microtiter plate readers and microtiter plate robotic systems. The present invention facilitates use of multilevel array systems that can be used in high-throughput screening (HTS), in the study of protein-protein interaction, cell based assays, colorimetric assays and other known biological or non-biological assays, as well as in many other low volume assay methods.
There is a need in the art for equipment which reduces the potential for errors and facilitates adding materials to receptacles such as microtiter plates or other multiwell plates for carrying out immunoassays, ligand binding assays, toxicity tests, inhibitor testing and PCR assays, especially qPCR assays.
The present inventors have solved this problem by creating a set of plate covers with openings (masks) such that each mask masks the wells for the three sample registers that are not in use for the current round of pre-mix dispensing. Each plate mask effectively converts a 384-well plate into a 96-well plate for that round of pre-mix dispensing. While the description of use for the set of plate masks described herein uses the example of qPCR, these masks could be used with SBS microtiter plates suitable for other applications such as cell culture, immunoassays, colorimetric assays, toxicity testing, as well as on higher density plates, such as the 1536-well format.
The need to reduce potential operator error with respect to dispensing into microtiter plates has been recognized and has led to the development of several devices, some of which are patented (See, for example, U.S. Pat. Nos. 4,919,894, 5,290,521 and 7,597,854). Prior efforts to decrease the likelihood of manual dispensing errors using microtiter plates have employed apparatus that are designed for many uses on many plates and which involve mechanical movable parts. Development of such apparatuses represents increasing complexity which, while providing advantages, also adds to the cost and complexity of manufacture and use. Use of the same apparatus with multiple microtiter plates present risk of cross-contamination between plates. In all assays such cross-contamination can be hard to detect and can compromise the validity of assay results. In the case of highly sensitive assays with high dynamic range such as those based on amplification, including PCR, this problem is accentuated. While it is possible to employ these apparatus only once or to thoroughly decontaminate the apparatus between uses, this approach imposes significant cost to the user. These apparatuses require that the positioning of a guide be changed between additions, and they do not provide for tactile or other feedback to the operator to provide confirmation of completion of addition of reagent to a set of wells. Provided herein is a solution that is simpler to manufacture and use, and which may provide feedback to the operator during use and owing to the simplicity of its manufacture, may be provided at a cost that makes single disposable use economically practical.
Commercially available devices, currently include the computer based devices WellAware™ from BioTX Automation, Inc. (Conroe, Tex., US; see the internet at biotx.net) and LightOne™ Illuminator FS from Embi Tec (San Diego, Calif., US; see the internet at embitec.com), the mechanically complex WelIMark® (U.S. Pat. No. 7,597,854) from Stovall Life Sciences, Inc. (Greensboro, N.C., US, and the sliding base and lid set called WellMatch from Gene Company Limited, (Hong Kong; see the internet at genehk.com). Additionally, none of these devices others of which the inventors are aware provide pipetting-action positive feedback of the addition of reagent to specific wells as described in some modes herein.